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Practical proteomics information

18 ul of sample will be injected per LC-MS run. Minimally 50 ul peptide solution (particle free, pH 2 to 4) has to be handed in. The detection limit for our nLC-MS system is better than 0.5 fmol. Please see our Proteomics Sample Preparation Protocol for details how to prepare samples.

Before every measuring series, the system [nLC (Proxeon EASY nLC) + MS (Thermo Orbitrap) + MSMS (Thermo LTQ-XL)] is checked by measuring a standard BSA digest. Points that are checked include LC peak width as well as MS and MSMS sensitivity. Also, no peptides from previous injections should be visual. When everything is alright, and only then, the next sample set will be injected with a “fast” cleaning gradient directly after each measurement gradient.

Gradient

Sample type

Number of samples per day

Standard gradient

In gel digests

10

Intermediate gradient

FASP samples low in hydrophobic peptides

6

Long gradient

FASP samples rich in hydrophobic peptides

4

When the standard gradient is used (generally used for samples obtained after an in-gel-digestion), the total measuring time is 2 hours per sample. Because of the checks before and some cleaning gradients after each sample set, 10 samples can be measured per day using the standard gradient. It is advised to put the Controls/Blancs first in your sample list, followed by the Samples. When the long gradient is used (e.g. for FASP samples), then maximally 4 samples can be measured in one day. When less than the maximal amount of samples is handed in, then the remaining time will be filled with cleaning gradients. Minimally one complete measurement day will therefore always be used and charged. When the system is so heavily contaminated that it cannot be cleaned within the same day (e.g. due to high concentrations of detergents) then an extra measurement day can be charged.

For identification and relative quantitation, the MaxQuant software package will be used. When a database is not publicly available, than a database in fasta format has to be handed in before measurements will be done. Search times necessary to compare the data to the database strongly depend both on the number of LCMS runs as well as on the database size. When a species specific database is used, it will take about 0.5 to 1 hour per run. The MaxQuant search result (a table with identification + normalized intensities) will be filtered with a filtering and statistics software called Perseus leaving confident identifications only. When applicable, a ratio graph will be added as well as significance info and e.g. hierarchical clustering when asked for. Significance info is only available for experiments done at least in triplicate. To be able to do it well, it is advised to do the experiments in fourfold. The Perseus filtered data as well as the original MaxQuant data [+ the protein abundance ratio Graph] will be supplied to you.

In the default MaxQuant graph, the Y-axis, usually named Log Total Intensity, is a summation of all the peak intensities measured of all identified peptides stemming from each protein. It is an indicator of the protein amount present in the samples.

The X-axis shows the ratio of the proteins peak intensities between each data set, usually named Sample and Control. In the example graph below, the majority of proteins do not vary in concentration between the Sample and Control data sets, resulting in a large cluster of proteins centered around a ratio of 1. These “non-variable” proteins often show up in a droplet shaped cluster since the variation between Sample and Control is (relatively) largest for low abundance proteins. 

In the graph it is easy to see that there are also proteins observed outside the droplet shape which actually are the main proteins of interest. For those proteins, on the right hand side of the droplet shaped “non-variable” proteins, the abundance in the sample is higher than in the control.

SjefMaxQuant.jpg

One remark. Since MaxQuant uses peak intensities for its calculations, chromatographic column overloading will result in relatively lower peaks for the most abundant proteins and therefore always have a ratio of 1.